Actinobacillus (formerly Haemophilus) pleuropneumoniae is a highly infectious porcine respiratory tract pathogen that causes porcine pleuropneumonia. Infected animals develop acute fibrinous pneumonia which leads to death or chronic lung lesions and reduced growth rates. Infection is transmitted by contact or aerosol and the morbidity in susceptible groups can approach 100%. Persistence of the pathogen in clinically healthy pigs also poses a constant threat of transmitting disease to previously uninfected herds.
The rapid onset and severity of the disease often causes losses before antibiotic therapy can become effective. Presently available vaccines are generally composed of chemically inactivated bacteria combined with oil adjuvants. However, whole cell bacterins and surface protein extracts often contain immunosuppressive components which make pigs more susceptible to infection. Furthermore, these vaccines may reduce mortality but do not reduce the number of chronic carriers in a herd.
There are at least 12 recognized serotypes of A. pleuropneumoniae with the most common in North America being serotypes 1, 5 and 7. Differences among serotypes generally coincide with variations in the electrophoretic mobility of outer membrane proteins and enzymes, thus indicating a clonal origin of isolates from the same serotype. This antigenic variety has made the development of a successful vaccination strategy difficult. Protection after parenteral immunization with a killed bacterin or cell free extract is generally serotype specific and does not prevent chronic or latent infection. Higgins, R., et al., Can. Vet. J. (1985) 26:86-89; MacInnes, J. I. and Rosendal, S., Infect. Immun. (1987) 55:1626-1634. Thus, it would be useful to develop vaccines which protect against both death and chronicity and do not have immunosuppressive properties. One method by which this may be accomplished is to develop subunit antigen vaccines composed of specific proteins in pure or semi-pure form.
An increasing number of bacterial antigens have now been identified as lipoproteins (Anderson, B. E., et al., J. Bacteriol. (1988) 170:4493-4500; Bricker, T. M., et al., Infect. Immun. (1988) 56:295-301; Hanson, M. S., and Hansen, E. J., Mol, Microbiol.(1991) 5:267-278; Hubbard, C. L., et al., Infect. Immun. (1991) 59:1521-1528; Nelson, M. B., et al., Infect. Immun. (1988) 56:128-134; Thirkell, D., et al., Infect. Immun. (1991) 59:781-784). One such lipoprotein from Haemophilus somnus has been positively identified. The nucleotide sequence for this lipoprotein, termed "LppA," has been determined (Theisen, M., et al., Infect. Immun. (1992) 60:826-831). These lipoproteins are generally localized in the envelope of the cell and are therefore exposed to the host's immune system. It has been shown that the murine lipoprotein from the outer membrane of Escherichia coli acts as a potent activator of murine lymphocytes, inducing both proliferation and immunoglobulin secretion (Bessler, W., et al., Z. Immun. (1977) 153:11-22; Melchers, F., et al., J. Exp. Med. (1975) 142:473-482). The active lipoprotein portion of the protein has been shown to reside in the N-terminal fatty acid containing region of the protein. Recent studies using synthetic lipopeptides based on this protein show that even short peptides, containing two to five amino acids covalently linked to palmitate, are able to activate murine lymphocytes (Bessler, W. G., et al., J. Immunol. (1985) 135:1900-1905).
It has been found that A. pleuropneumoniae possesses several outer membrane proteins which are expressed only under iron limiting growth conditions (Deneer, H. G., and Potter, A. A., Infect. Immun. (1989) 57:798-804). However, outer membrane lipoproteins from A. pleuropneumoniae have not heretofore been identified or characterized with respect to their immunogenic or protective capacity.